1. Field of the Invention
The present invention relates generally to the fields of protein biochemistry and medicine. More particularly, it concerns bioactive, low-toxicity fragments of protamine, and a number of different uses of such protamine fragment compositions. Provided are protamine fragments, compositions, combinations and kits and various methods and uses of such fragments, e.g., in the neutralization of heparin and for association with a variety of therapeutic proteins, including insulin.
2. Description of Related Art
Heparin has become the clinical anticoagulant drug of choice, and is used universally for: prophylaxis of postoperative thromboembolism, in patients with stroke, during various surgical situations, and particularly in all procedures involving extracorporeal blood circulation (ECBC) (Jaques, 1980; Majerus et al., 1996). Extracorporeal blood circulation is employed in numerous clinical situations such as kidney dialysis, open-heart operations, cardiac catheterizations, blood oxygenation, plasmapheresis, organ transplantation, and the implantation of artificial organs. In the United States alone, approximately 15 million ECBC procedures are performed annually (Ma et al., 1994). Approximately 33 metric tons of heparin, representing 500 million doses, are used worldwide each year (Linhardt, 1991). The production figures obtained from the pharmaceutical industry suggest that clinical use of heparin continues to grow rapidly (Linhardt, 1991).
Heparin consists of a heterogeneous mixture of sulfated glycosaminoglycans with a molecular weight ranging from 3,000 to 40,000 daltons. It is made of a repeating unit of D-glucuronic acid and N-acetyl-D-glucosamine residues (Bourin and Lindahl, 1993). The anticoagulant function of heparin was discovered over 70 years ago (Howell, 1922). Heparin exerts its anticoagulant activity primarily via interaction with antithrombin III (Rosenberg, 1987).
Antithrombin III (ATIII) is a circulating inhibitor of the serine proteases in the coagulation cascade, acting more particularly on thrombin and factor Xa but also on factors IXa, XIa, and XIIa. It possesses an arginine center that binds to the active serine site of thrombin (and also the other coagulation factors) to form a covalent bond (Griffith, 1983). Normally this reaction proceeds rather slowly. Binding of heparin to ATIII, however, induces a conformational change of ATIII, rendering the arginine center more accessible to thrombin interaction, and producing a 1000-fold acceleration of the inhibitory effect (Rosenberg, 1987). The binding of heparin to ATIII involves a unique pentasaccharide sequence containing a 3-O-sulfated glucosamine residue (Choay et al., 1981), and entails interaction between specific lysine residues on ATIII and sulfate and carboxylate groups in heparin (Choay et al., 1981; Rosenberg et al., 1979).
Heparin also acts as a template that helps bring thrombin in close proximity to ATIII. Thrombin then cleaves the reactive site bond of ATIII, to which it becomes covalently bound and is irreversibly inhibited. The released heparin can then act on other ATIII molecules. Heparin, however, does not act as a template for the interaction of antithrombin III and factor Xa (Casu et al., 1981). Thus smaller heparin fragments, such as the low molecular weight heparin possessing the ATIII-binding sequence, are able to inhibit factor Xa but not thrombin (Verstraete, 1990).
Systemic heparinization, however, results in a high incidence of bleeding complications (Hirsh, 1984; Kelton and Hirsh, 1984). Major bleeding occurs in 8% to 33% of patients who receive various forms of heparin therapy (Levin and Hirsh, 1986). Nearly 25% of all patients suffering from acute renal failure are subject to increased bleeding risk during and immediately following dialysis (Swartz and Port, 1979). The incidence of bleeding increase with elderly or diabetic patients, patients with ulcers or other multiple traumata, and patients with current cardiac or vascular surgery.
Aside from hemorrhage, there are also other complications associated with the use of heparin, particularly when the drug is administered over a long period. These added complications include thrombocytopenia, alopecia, arterial embolus, and interference with bone repair and maintenance (Hirchboeck et al., 1954). In fact, heparin has been cited as xe2x80x9cthe drug responsible for the most deaths in patients who are reasonably healthyxe2x80x9d (Porter and Jick, 1977).
Low molecular weight heparin (LMWH) was derived from native heparin in an attempt to abate the induced bleeding risk (Verstraete, 1990; Holmer et al., 1986). It contains the specific pentasaccharide sequence in heparin that is required for ATIII binding, and thus fully retains the antithrombotic effect of heparin through inhibition of factor Xa by ATIII. However, it is of insufficient length to bind thrombin and catalyze the inhibition of thrombin by ATIII. LMWH is more effective than regular heparin in preventing deep vein thrombosis and pulmonary embolism after orthopedic surgery but has similar incidence of bleeding (Jensen and Ens, 1993). In a large randomized study in which a continuous intravenous infusion of heparin was compared with a fixed subcutaneous dose of LMWH in patients with venous thrombosis, the incidence of major bleeding was only marginally lower with LMWH (Hull et al., 1992). Enoxparin (Clexane), Kabi-2165 (Fragmin), CY-216 (Fraxiparine), and Novo LHN-1 (Logiparin) are a few commercial LMWH products approved for clinical use.
A major drawback of LMWH lies in the absence of an appropriate clinical antidote to combat the potential risk of induced bleeding. Neither protamine nor platelet factor 4 (PF4, a naturally occurring protein from platelet that is under extensive investigation as a potential replacement of protamine as the anti-heparin agent (Cook et al., 1992) can fully neutralize the anticoagulant effects of LMWH (Lechner et al., 1995; Ryn-McKenna et al., 1990).
To reduce post-operative bleeding, protamine, a clinical heparin antagonist, is routinely administered after cardiac and vascular surgery to reverse the anticoagulant activity of heparin (Jaques, 1973). Protamine consists of a group of heterogeneous polycationic peptides with an average molecular weight of about 4500 daltons. It is generally obtained from fish (Ando et al., 1973). Nearly 67% of the amino acid composition of protamine is arginine (Ando et al., 1973). The polycationic protamine combines electrostatically with the polyanionic heparin to form a stable complex that is devoid of anticoagulant activity. Each milligram of protamine neutralizes approximately 90 units of heparin derived from bovine lung tissue or 115 units of heparin derived from porcine intestinal mucosa. Protamine, however, cannot completely neutralize the anticoagulant activities of low molecular weight heparins (Lechner et al., 1995; Ryn-McKenna et al., 1990; Harenberg et al., 1985; Diness and Ostergaard, 1986; Wakefield et al., 1994), apparently due to an insufficient binding affinity between protamine and LMWH.
In addition to its function as a heparin antagonist, protamine also finds another major pharmacological application. It prolongs the adsorption of insulin, and is therefore combined with insulin to formulate protamine zinc insulin (PZI) and neutral protamine Hagedorn (NPH) insulin. Such formulations allow insulin-dependent diabetic patients to achieve euglycemia with less frequent insulin injections.
However, despite its nearly universal use in clinical practice, current formulations of protamine are nevertheless toxic. Protamine toxicity ranges from mild hypotension (Katz et al., 1987; Ovrum et al., 1992; Kirklin et al., 1986), to severe systemic vascular collapse requiring prompt intervention (Lowenstein et al., 1983; Just-Viera et al., 1984; Hurby et al., 1995), or idiosyncratic fatal cardiac arrest (Olinger et al., 1980; Cobb and Fung, 1982; Sharath et al., 1985; Neidhart et al., 1992).
The protamine toxicity is mediated by several pathways: (i) non-immunological pathway; (ii) immunoglobulin-mediated pathway; (iii) inhibition of carboxypeptidase N; and (iv) other toxic effects. Anaphylactoid type of reactions produced via the first mechanism, which are manifested by complement activation, thromboxane generation, and histamine release, are more common and less dangerous. Anaphylactic types of responses produced via the second pathway, however, are unpredictable, not preventable, and always life-threatening. More than 100 deaths have been attributed to this type of protamine toxicity (Horrow, 1985; Lindblad, 1989). Thus, protamine compositions without any one or more of these or other toxic effects, and the use of such compositions in place of protamine, would represent a significant advance in the art.
The present invention overcomes one or more of these and other shortcomings in the art by providing bioactive protamines and compositions, combinations and kits thereof, in which the protamines have reduced immunogenicity, antigenicity and/or toxicity compared to native protamine. Preferably, the bioactive, low-toxicity protamines and compositions thereof are low molecular weight protamines. Various methods and uses of such low molecular weight bioactive protamines, compositions, combinations and kits are also provided, including antagonizing heparin functions to reduce post-operative bleeding and prolonging the adsorption of insulin to treat diabetes.
The invention thus provides purified protamines that are bioactive, that have reduced immunoresponsiveness and/or toxicity compared to native protamine and that preferably have a low molecular weight. The terms xe2x80x9cprotaminexe2x80x9d, xe2x80x9cprotamine speciesxe2x80x9d, xe2x80x9cprotamine polypeptidexe2x80x9d, xe2x80x9cprotamine peptidexe2x80x9d and xe2x80x9cprotamine fragmentxe2x80x9d are generally used interchangeably and, unless otherwise specifically stated, each refer to the unique low molecular weight protamines of the invention. Also, unless otherwise specifically stated, the protamines, compositions, combinations, kits, methods and uses of the invention encompass single, highly purified low molecular weight protamine species and more than one, or a plurality of, low molecular weight protamine species.
In providing purified, bioactive protamine species that have reduced toxicity compared to native protamine, the xe2x80x9creduction in toxicityxe2x80x9d includes reductions in toxicity that are mediated by any one or more of several mechanisms, including non-immunological pathways, immunoglobulin-mediated pathways, inhibition of carboxypeptidase N, and other toxic effects. xe2x80x9cReductions in toxicityxe2x80x9d thus include reductions in anaphylactoid reactions, complement activation, thromboxane generation and histamine release. Clinically, xe2x80x9creductions in toxicityxe2x80x9d include reductions in hypotension, systemic vascular collapse and/or idiosyncratic fatal cardiac arrest.
In providing purified protamine species that are bioactive and have reduced immunoresponsiveness, the term xe2x80x9creduced immunoresponsivenessxe2x80x9d includes reduced immunogenicity (reduced ability to induce an immune response in an animal in vivo, including T cell and B cell responses, as well as antibody production) and/or reduced antigenicity (reduced ability to be recognized by anti-protamine antibodies) compared to native protamine.
The various purified, bioactive, low-toxicity protamine species of the invention thus have only about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, about 0.005%, about 0.001% or less of the immunoresponsiveness and/or toxicity, including low molecular weight protamine species that have no immunoresponsiveness and/or toxicity, within detection limits, compared to native protamine.
Preferably, the purified, bioactive, low-toxicity protamine species will have substantially reduced immunoresponsiveness and/or toxicity compared to native protamine. The term xe2x80x9csubstantially reduced immunoresponsiveness and/or toxicityxe2x80x9d means that the purified, bioactive, low-toxicity protamine species preferably have only about 50% of the immunoresponsiveness and/or toxicity of native protamine, or more preferably about 30% of the imnmunoresponsiveness and/or toxicity of native protamine, or even more preferably have only about 10% or 5% or less of the immunoresponsiveness and/or toxicity of native protamine. Protamine species with only about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, about 0.005%, about 0.001% and no detectable imnmunoresponsiveness and/or toxicity, compared to native protamine, are naturally included as purified, bioactive, low-toxicity protamines with xe2x80x9csubstantially reduced immunoresponsiveness and/or toxicity compared to native protaminexe2x80x9d.
As used herein, the term xe2x80x9cnative protaminexe2x80x9d refers to protamine in the state that it is originally isolated in, that is, protamine that includes intact, high molecular weight species. This will be understood to include presently commercially available protamine preparations, as well as protamine isolated from natural sources, such as sperm heads and/or fertilized eggs.
Unless otherwise specifically stated, the purified, bioactive, low-toxicity protamine, protamines, compositions, combinations, kits, methods and uses of the invention encompass single, highly purified, bioactive, low-toxicity protamine species and more than one, or a plurality of, purified, bioactive, low-toxicity protamine species. The invention therefore includes one, two, three, four, five, six, seven, eight, nine or ten or more, distinct, bioactive, low-toxicity protamine species, as well as populations, sub-populations and pluralities thereof. Preferably, xe2x80x9cpurifiedxe2x80x9d bioactive, low-toxicity protamines are also low molecular weight protamines, such that xe2x80x9cpurifiedxe2x80x9d refers to the purification of one or more distinct low molecular weight protamine species xe2x80x9caway from high molecular weight speciesxe2x80x9d. The single, multiple or pluralities of protamine species of the invention are thus those that have been isolated away from, purified free from, or in certain aspects, isolated substantially away from or purified substantially free from, high molecular weight protamine species.
The purified, low-toxicity, preferably low molecular weight, protamine species of the present invention are xe2x80x9cbioactivexe2x80x9d, that is, they retain sufficient bioactivity for any practical purpose. The terms xe2x80x9cbioactivexe2x80x9d and xe2x80x9csufficient bioactivityxe2x80x9d mean that the low-toxicity protamines of the invention are protamine species that at least maintain or retain one, some, most or all of the biological activities of xe2x80x9cnative protaminexe2x80x9d.
The biological activities of native protamine include, but are not limited to, the ability to at least partially bind to and/or neutralize heparin and/or low molecular weight heparin and the ability to associate with, or form a complex with, certain therapeutic proteins or peptides, such as insulin or xcex1-interferon. The ability to bind to and/or neutralize low molecular weight heparin is a preferred property of the purified, bioactive, low-toxicity protamines of the present invention. However, the binding properties are not limited to heparin and, for example, may extend to heparan, heparan sulfate and the like.
In various aspects of the present invention, the bioactive protamines will include at least a first protamine species that has at least about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or even up to at least about 100% of the heparin or low molecular weight heparin binding and/or neutralizing capability of native protamine. Thus, the present invention provides at least a first purified protamine species, polypeptide or peptide, preferably a low molecular weight protamine, that retains heparin binding and/or neutralization activity and that has reduced immunoresponsiveness and/or toxicity compared to native protamine.
Preferably, the purified, low-toxicity protamine species maintain or retain significant or substantial bioactivity compared to native protamine. The term xe2x80x9csignificant or substantial bioactivityxe2x80x9d means that the purified protamine species preferably maintain or retain at least about 20% of the bioactivity of native protamine, more preferably at least about 40% of the bioactivity of native protamine, and even more preferably maintain or retain at least about 60% or 80% or more of the bioactivity of native protamine.
In that native protamine does not sufficiently bind low molecular weight heparins and cannot completely neutralize the anticoagulant activities of low molecular weight heparins, the present invention is able to provide protamine species that have xe2x80x9cat leastxe2x80x9d about 100% of the heparin or low molecular weight heparin binding and/or neutralizing capability of native protamine. Thus, the low-toxicity protamine species of the invention extend to species that have xe2x80x9cmorexe2x80x9d low molecular weight heparin binding and/or neutralizing capability than native protamine. These include purified protamine species that have xe2x80x9cincreased bioactivityxe2x80x9d compared to native protamine.
Furthermore, it will be understood that preferred purified low-toxicity protamine species will be those that combine preferable levels of bioactivity and acceptably reduced levels of immunoresponsiveness and/or toxicity. Additionally, these characteristics are related, such that for purified protamine species that have dramatically reduced immunoresponsiveness and/or toxicity, for example 10% or 5% or less of the immunoresponsiveness and/or toxicity of native protamine, less substantial bioactivity, for example even 5% or 10% or so would be preferable in certain aspects of the invention. Likewise, for purified protamine species that have less dramatically reduced immunoresponsiveness and/or toxicity, for example 90% or 80% or so of the immunoresponsiveness and/or toxicity of native protamine, more substantial bioactivity, for example 70% or 80% or so would be more preferable in certain aspects of the invention.
The bioactive protamine, protamines, compositions, combinations, kits, methods and uses of the invention generally comprise at least a first purified low molecular weight bioactive protamine that has a molecular weight of between about 400 and about 3000 daltons or so. In certain aspects of the invention, the at least a first purified low molecular weight bioactive protamine has a molecular weight of between about 400 and about 2500 daltons, about 400 and about 2000 daltons, about 400 and about 1500 daltons, about 400 and about 1400 daltons, about 400 and about 1350 daltons, about 400 and about 1300 daltons, about 400 and about 1200 daltons, about 400 and about 1100 daltons, about 400 and about 1000 daltons. In further aspects, the at least a first purified low molecular weight bioactive protamine has a molecular weight of between about 500 and about 3000 daltons, about 600 and about 3000 daltons, about 700 and about 3000 daltons, about 800 and about 3000 daltons, about 900 and about 3000 daltons, about 1000 and about 3000 daltons, about 1500 and about 3000 daltons. In still further aspects, the at least a first purified low molecular weight bioactive protamine has a molecular weight of between about 2000 and about 3000 daltons, about 500 and about 2500 daltons, about 1000 and about 2000 daltons, or about 1000 and about 1500 daltons.
In certain preferred embodiments, the composition comprises at least a first purified low molecular weight bioactive protamine that has a molecular weight of between about 1100 and about 1350 daltons, preferably of between about 1150 and about 1300 daltons.
In further aspects of the present invention, the protamine, protamines, compositions, combinations, kits, methods and uses of the invention comprises at least a first purified low molecular weight bioactive protamine that has a molecular weight of about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, -about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900 or about 3000 daltons.
In currently preferred embodiments, the composition comprises at least a first purified low molecular weight bioactive protamine that has a molecular weight of about 1200 daltons. xe2x80x9cAbout 1200 daltonsxe2x80x9d includes 1160, 1170, 1180, 1190, 1195, 1205, 1210, 1220, 1230, 1240 daltons and such like,
Defined aspects of the invention are thus compositions comprising at least a first purified, low-toxicity, bioactive protamine. Such compositions may comprise only a first purified bioactive protamine species, or at least a first and at least a second purified bioactive protamine species. In yet other aspects, these compositions further comprise at least a third, at least a fourth, at least a fifth, at least a sixth, at least a seventh, at least an eighth, at least a ninth or at least a tenth purified bioactive protamine species, polypeptide and/or peptide. In certain embodiments, the composition comprises a population, sub-population or plurality of purified bioactive protamine species, polypeptides and/or peptides that have reduced immunogenicity, antigenicity and/or toxicity compared to native protamine.
The population, sub-population or plurality of low-toxicity protamine species can each have similar, or in other aspects, distinct molecular weights within the above stated ranges. Thus, in certain embodiments, the compositions comprise a plurality of purified bioactive protamine species, polypeptides and/or peptides that have a molecular weight of between about 400 and about 2500 daltons, while in further embodiments, the compositions comprise a plurality of purified bioactive protamine species, polypeptides and/or peptides that have a molecular weight of about 1200 daltons.
Any source of protamine is contemplated for use in the preparation of the instant protamine compositions, combinations, kits, methods and uses. Preferred sources of protamine include, but are not limited to, mammals, amphibians and fish, with salmon and herring being more preferred in certain aspects of the invention. Thus, in particular embodiments, the compositions, combinations, kits, methods and uses comprise at least a first purified bioactive salmine (salmon) protamine and/or at least a first purified bioactive clupeine (herring) protamine.
In certain embodiments of the present invention, the purified, bioactive protamine compositions will further comprise at least a first additional (distinct) biologically active agent. The term xe2x80x9cat least a first additional or distinct biologically active agentxe2x80x9d, as it applies to the compositions, combinations, kits, methods and uses of the invention means at least a first biologically active agent xe2x80x9cin addition toxe2x80x9d the at least a first purified bioactive protamine species. Accordingly, the at least xe2x80x9ca first additionalxe2x80x9d biologically active agent could well be termed at least xe2x80x9ca second, distinctxe2x80x9d biologically active agent, with the xe2x80x9cat least a first purified bioactive protaminexe2x80x9d being at least a first biologically active agent. However, this is purely a matter of semantics, and the practical meaning will be clear to those of ordinary skill in the art.
In exemplary embodiments, the low-toxicity, preferably low molecular weight, bioactive protamine-containing compositions, combinations, kits, methods and uses of the invention will further comprise at least one additional biologically active agent in the form of at least a second coagulant, coagulation factor or coagulative, thrombotic or clot-inducing therapeutic agent. A plurality of coagulants, coagulation factors or coagulative, thrombotic or clot-inducing therapeutic agents may also be employed.
Exemplary coagulation factors for such uses include Tissue Factor and Tissue Factor derivatives, including truncated, mutant, dimeric, polymeric and multimeric Tissue Factor derivatives (WO 96/01653; specifically incorporated herein by reference); one or more of the vitamin K-dependent coagulation factors, preferably Factor II/IIa, Factor VII/VIIa, Factor IX/IXa and/or Factor X/Xa; and other coagulation factors such as Factor V/Va, VIII/VIIIa, Factor XI/XIa, Factor XII/XIIa and Factor XIII/XIIIa. Further suitable coagulation factors are Russell""s viper venom Factor X activator; platelet-activating compounds, such as thromboxane A2 and thromboxane A2 synthase; and inhibitors of fibrinolysis, such as xcex12-antiplasmin.
Further biologically active agents include therapeutic proteins to be delivered to an animal or patient. Preferred therapeutic proteins are generally those that have an acidic pKa value are preferred for use with the low molecular weight protamine compositions. The biologically active proteins for use in combination with the compositions, combinations, kits, methods and uses of the invention include, but are not limited to, insulin (including recombinant insulin), preferably human insulin, and xcex1-interferon.
The present invention also provides pharmaceutical compositions comprising a biologically effective amount of at least a first purified bioactive protamine that has reduced immunogenicity, antigenicity and/or toxicity compared to native protamine, and a pharmaceutically acceptable diluent, excipient or carrier. Injectable pharmaceutical compositions exemplify the pharmaceuticals of the invention. In other aspects, the pharmaceutical composition further comprises a biologically effective amount of at least a first biologically active agent, such as a coagulant or a protein with an acidic pKa value.
In certain embodiments, the pharmaceutical composition further comprises a biologically effective amount of insulin, human insulin and/or xcex1-interferon. The invention thus provides pharmaceutical compositions comprising a combined effective amount of at least a first purified bioactive, low-immunoresponsive and/or low-toxicity protamine and insulin, formulated in a pharmaceutically acceptable diluent or vehicle.
Also provided are kits comprising, in at least a first suitable container, a composition comprising one or more purified bioactive, low-immunoresponsive and/or low-toxicity protamines. The kits may also be combination kits comprising, in at least a first suitable container, a composition comprising at least a first purified bioactive, low-immunoresponsive and/or low-toxicity protamine and at least a first biologically active agent, such as a coagulant or a protein with an acidic pKa value. Biologically effective amounts of insulin, human insulin and/or xcex1-interferon are currently preferred. The protamines and other biologically active agent(s) of the kits may be formulated in a single container or in at least two distinct containers.
Still further kits of the invention are those that comprise at least a first suitable container comprising at least a first anticoagulant; and at least a second suitable container comprising at least a first purified bioactive protamine in accordance with the present invention. Currently preferred anticoagulants for use in such kits are heparin and low molecular weight heparin. These kits may also comprise further biologically active agents, such as additional anticoagulants, additional coagulants, proteins, insulin, xcex1-interferon and the like.
The purified bioactive protamines, compositions, combinations and kits of the invention have uses in binding, and preferably inhibiting and/or inactivating, heparin and/or low molecular weight heparin in vitro and in vivo; have uses as heparin antagonists and/or low molecular weight heparin antagonists in vitro and in vivo; have uses in ameliorating and/or reversing the anticoagulant activity of heparin and/or low molecular weight heparin in vitro and in vivo; and have uses in preventing or reducing pre-, mid- and/or post-operative bleeding, such as when following heparin and/or low molecular weight heparin administration to an animal or human subject.
Where the purified bioactive protamines and compositions of the invention have been combined with biologically active agents, proteins with acidic pKa values, insulin and/or xcex1-interferon, the resultant compositions and kits of the invention have uses in prolonging the adsorption and/or the bioavailability of the biologically active agents, proteins with acidic pa values, insulin and/or xcex1-interferon upon administration to an animal or human subject. Purified bioactive protamine compositions and kits in combination with insulin are particularly intended for use in treating diabetes in an animal or human subject.
The invention thus further provides for the use of a purified bioactive protamine composition, combination and/or kit in the manufacture of a medicament for use in treating post-operative bleeding in an animal or human subject. The invention still further provides for the use of a purified bioactive protamine-insulin composition or combination, and/or kit, in the manufacture of a medicament for use in treating diabetes in an animal or human subject.
The present invention further provides methods of preparing at least a first bioactive, preferably low molecular weight, protamine or protamine fraction that has reduced immunoresponsiveness and/or toxicity compared to native protamine (or a plurality or population of such protamines or protamine fractions). The methods generally comprise contacting a native protamine composition with at least a first proteolytic composition comprising at least a first proteolytic enzyme in an amount and for a period of time effective to produce a low molecular weight bioactive protamine, protamine fraction or a plurality or population thereof.
The types and amounts of the proteolytic compositions and enzymes, and the times of incubation, exposure or contact, can be used to prepare low molecular weight bioactive protamine species, polypeptides or peptides that have any of the molecular weights referenced herein, as exemplified by those of between about 450 daltons and about 2500 daltons; between about 450 daltons and about 1350 daltons; and of about 1200 daltons or so.
In certain aspects of the invention, the at least a first proteolytic enzyme is removed from the admixture after the low molecular weight bioactive protamine(s) is produced. The methods thus further comprise isolating the at least a first low molecular weight protamine polypeptide thus produced. Accordingly, the invention encompasses the at least a first low molecular weight protamine polypeptide(s) produced. The at least a first low molecular weight bioactive protamine(s) so produced may also be formulated in a pharmaceutically acceptable composition, combination or kit.
Proteolytic enzymes contemplated for use in the production of the low molecular weight bioactive protamine species, polypeptides and/or peptides include, but are not limited to, thermolysin, ficin, collagenase, kallikrein, proline-specific endopeptidase, or any combination thereof. In other embodiments, the methods comprise contacting the native protamine composition with at least a first and at least a second proteolytic enzyme.
The invention further provides methods of selecting an improved low molecular weight protamine polypeptide, species or fraction, comprising selecting from a plurality of low molecular weight protamine polypeptides, species or fractions a low molecular weight protamine polypeptide, species or fraction that substantially retains the bioactivity of native protamine and has substantially reduced immunoresponsiveness and/or toxicity as compared to native protamine.
The methods of selecting an improved low molecular weight protamine polypeptide, species or fraction generally comprise preparing a population of low molecular weight protamine polypeptides, species or fractions and selecting from the population of low molecular weight protamine polypeptides, species or fractions a low molecular weight protamine polypeptide, species or fraction that retains substantial bioactivity and has substantially reduced immunoresponsiveness and/or toxicity compared to native protamine. The population of low molecular weight protamine polypeptides, species or fractions are preferably generated by contacting a native protamine composition with at least a first proteolytic composition comprising at least a first proteolytic enzyme, as described above.
In certain embodiments, the population of low molecular weight protamine polypeptides is first selected for bioactivity, and then for reduced immunoresponsiveness. In other embodiments, the population of low molecular weight protamine polypeptides is first selected for reduced immunoresponsiveness, and then for bioactivity. These properties may be balanced and off-set to yield a range of improved low molecular weight protamines.
In certain aspects of these methods, any proteolytic enzyme is removed after the improved low molecular weight bioactive protamine(s) is identified. The methods thus further comprise isolating the improved low molecular weight protamine polypeptide so identified. Accordingly, the invention encompasses the improved low molecular weight protamine polypeptide(s) identified. The improved low molecular weight bioactive protamine(s) so identified may also be formulated in a pharmaceutically acceptable composition, combination or kit.
Also provided by the present invention are methods of inhibiting or inactivating heparin or low molecular weight heparin, comprising contacting heparin or low molecular weight heparin with an effective amount of at least a first purified, low-toxicity bioactive protamine in accordance with the present invention.
In certain preferred embodiments, the heparin or low molecular weight heparin is located within a mammal and the composition is administered to the mammal. In particular aspects of the invention, the heparin or low molecular weight heparin is contacted with a combined effective amount of the composition comprising at least a first purified bioactive protamine and at least a second, distinct agent, such as a coagulant, a protein with an acidic pKa value, insulin and/or xcex1-interferon.
The invention further provides methods of ameliorating or reducing an effect of heparin in a mammal, comprising administering to the mammal a therapeutically effective amount of at least a first pharmaceutical composition comprising at least a first purified bioactive protamine that has reduced immunoresponsiveness and/or toxicity compared to native protamine. In certain aspects, the mammal has diabetes and/or is receiving injections of an insulin-protamine complex.
The invention also provides methods of treating or preventing excessive bleeding in a mammal, comprising administering to the mammal a therapeutically effective amount of at least a first pharmaceutical composition comprising at least a first purified bioactive protamine that has reduced immunoresponsiveness and/or toxicity compared to native protamine. In certain aspects, at least a second coagulant or coagulative, thrombotic or clot-inducing therapeutic agent is further administered to the mammal. In certain aspects, the mammal has diabetes and/or is receiving injections of an insulin-protamine complex. The excessive bleeding can be associated with a number of different conditions, including, but not limited to, systemic heparinization, extracorporeal blood circulation, one or more particular diseases or disorders, a trauma or with surgery.
The instant bioactive protamine compositions, combinations and kits also find use in prolonging the bioavailability of various biologically active or therapeutic proteins and/or peptides. Thus, the present invention provides methods of prolonging the bioavailability of a protein with an acidic pKa value, such as insulin, human insulin or xcex1-interferon upon administration to a mammal, comprising co-administering the protein with an acidic pKa value, insulin or xcex1-interferon to the mammal in combination with an effective amount of at least a first purified bioactive protamine that has reduced immunoresponsiveness and/or toxicity compared to native protamine.
Also provided by the present invention are methods of treating or preventing diabetes in a mammal, comprising administering to the mammal a therapeutically effective amount of insulin and at least a first purified bioactive protamine that has reduced immunoresponsiveness and/or toxicity compared to native protamine. The insulin and the at least a first purified bioactive protamine may be administered in a single pharmaceutical composition or in distinct pharmaceutical compositions administered within a biologically effective time.
In all uses and methodological aspects of the present invention, the mammals to be treated include human subjects.